[Histonet] TRAP Staining Protocol

[Herrick, James L. (Jim)]

Hello everyone,

I have a question regarding a good TRAP stain protocol. We have pretty good luck using our protocol on human iliac crest biopsies, but the stain tends to fade to almost nothing overnight, when we use it on rat and mouse femurs, vertebrae, etc.. We do see some fading in our iliac crest biopsies, but it is not as severe as the animal specimens.

Our specimens are embedded in GMA or MMA and the constituents of our protocol consist of pararosanilin (Basic Red), Acetate Buffer (solutions A & B), Naphthol AS-TR Phosphate and Manganese Chloride. We incubate at 37°C for 60 to 120 minutes, rinse with dH2O, dehydrate through graded alcohols and coverslip with Eukitt mounting medium. Does anyone know why our stains are fading or would you possibly have a different protocol that we can try? Any suggestions you may have would be greatly appreciated. Thanks for your help.

Jim